Amylin Family Polypeptide-6 (AFP-6) is a member of the Amylin Family, which includes amylin, adrenomedullin (ADM), calcitonin (CT), and calcitonin gene related peptide (CGRP). The human AFP-6 gene, also known as intermedin, encodes a 148 amino acid open reading frame with a 24 amino acid signal peptide for secretion at the N-terminus and a mature amidated peptide having an amino acid sequence of
(SEQ ID NO: 1)TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY- NH2.
Other AFP-6 polypeptides include:
(SEQ ID NO: 2)VGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY-NH2; (SEQ ID NO: 3)PHAQLLRVGCVLGTCQVQNLSHRLWQLVRPAGRRDSAPVDPSSPHSY- NH2;and (SEQ ID NO: 4)VGCVLGTCQVQNLSHRLWQLVRPAGRRDSAPVDPSSPHSY-NH2.Still other Amylin Family polypeptides include:
(SEQ ID NO: 5)KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH2; (SEQ ID NO: 6)CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-NH2; (SEQ ID NO: 7)ACDTATCVTHRLAGLLSRSGGWKNNFVPTNVGSKAF-NH2; (SEQ ID NO: 8)ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-NH2; (SEQ ID NO: 9)YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQ GY-NH2.
Table 1 shows the polypeptide corresponding to the SEQ ID NOs. shown above. SEQ ID NOs: 1-4 relate to forms of human and mouse AFP-6. SEQ ID NOs: 5-9 relate to human Amylin Family polypeptides of amylin, calcitonin, CGRP α, CGRP β, and adrenomedullin.
TABLE 1SEQ ID NO: 1Human AFP-6 [1-47]SEQ ID NO: 2Human AFP-6 [8-47]SEQ ID NO: 3Mouse AFP-6 [1-47]SEQ ID NO: 4Mouse AFP-6 [8-47]SEQ ID NO: 5Human AmylinSEQ ID NO: 6Human CalcitoninSEQ ID NO: 7Human CGRP αSEQ ID NO: 8Human CGRP βSEQ ID NO: 9Human Adrenomedullin
It has been reported that the biological actions of these Amylin Family polypeptides are mediated via binding to two closely related type II G protein-coupled receptors (GPCRs), the calcitonin receptor (CTR) and the calcitonin receptor like receptor (CRLR). Cloning and functional studies have shown that CGRP, ADM, and amylin interact with different combinations of CTR or the CRLR and the receptor activity modifying protein (RAMP). Many cells express multiple RAMPs. It is believed that co-expression of RAMPs and either the CTR or CRLR is required to generate functional receptors for calcitonin, CGRP, ADM, and amylin. The RAMP family comprises three members (RAMP1, -2, and -3), which share less then 30% sequence identity, but have a common topological organization. Co-expression of CRLR and RAMP 1 leads to the formation of a receptor for CGRP. Co-expression of CRLR and RAMP2 leads to the formation of a receptor for ADM. Co-expression of CRLR and RAMP3 leads to the formation of a receptor for ADM and CGRP. Co-expression of hCTR2 and RAMP1 leads to the formation of a receptor for amylin and CGRP. Co-expression of hCTR2 and RAMP3 leads to the formation of a receptor for amylin.
Amylin regulates gastric emptying and suppresses glucagon secretion and food intake, thus regulating the rate of glucose appearance in the circulation. It appears to complement the actions of insulin, which regulates the rate of glucose disappearance from the circulation and its uptake by peripheral tissues. These actions are supported by experimental findings in rodents and humans, which indicate that amylin complements the effects of insulin in postprandial glucose control by at least three independent mechanisms, all of which affect the rate of glucose appearance. First, amylin suppresses postprandial glucagon secretion. Compared to healthy adults, patients with type 1 diabetes have no circulating amylin and patients with type 2 diabetes have diminished postprandial amylin concentrations. Furthermore, infusion of an amylin specific monoclonal antibody, which bound circulating amylin, again resulted in greatly elevated glucagon concentrations relative to controls. Both of these results point to a physiological role of endogenous amylin in the regulation of postprandial glucagon secretion. Second, amylin slows gastrointestinal motility and gastric emptying. Finally, intrahypothalamic injections of rat amylin were shown to reduce feeding in rats and alter neurotransmitter metabolism in the hypothalamus. In certain studies, food intake was significantly reduced for up to eight hours following the intrahypothalamic injection of rat amylin and rat CGRP. In human trials, an amylin analog, pramlintide, has been shown to reduce weight or weight gain. Amylin may be beneficial in treating metabolic conditions such as diabetes and obesity. Amylin may also be used to treat pain, bone disorders, gastritis, to modulate lipids, in particular triglycerides, or to affect body composition such as the preferential loss of fat and sparing of lean tissue.
The hormone calcitonin (CT) was named for its secretion in response to induced hypercalcemia and its rapid hypocalcemic effect. It is produced in and secreted from neuroendocrine cells in the thyroid that have since been termed C cells. The best-studied action of CT(1-32) is its effect on the osteoclast. In vitro effects of CT include the rapid loss of ruffled borders and decreased release of lysosomal enzymes. Ultimately, the inhibition of osteoclast functions by CT results in a decrease in bone resorption. However, neither a chronic reduction of serum CT in the case of thyroidectomy nor the increased serum CT found in medullary thyroid cancer appears to be associated with changes in serum calcium or bone mass. It is thus most likely that a major function of CT(1-32) is to combat acute hypercalcemia in emergency situations and/or protect the skeleton during periods of “calcium stress” such as growth, pregnancy, and lactation. Reviewed, for example, in Becker (2004) JCEM 89(4):1512-1525 and Sexton (1999) Current Medicinal Chemistry 6:1067-1093. Consistent with this is recent data from the calcitonin gene knockout mouse, which removes both the calcitonin and the CGRP-I peptides, that revealed that the mouse had normal levels of basal calcium-related values, but an increased calcemic response (Kurihara et al. (2003) Hypertens Res. 26 Suppl:S 105-108).
CT has an effect on plasma calcium levels and inhibits osteoclast function and is widely used for the treatment of osteoporosis. Therapeutically, salmon CT appears to increase bone density and decrease fracture rates with minimal adverse effects. CT has also been successfully used over the past 25 years as a therapy for Paget's disease of bone, which is a chronic skeletal disorder that may result in enlarged or deformed bones in one or more regions of the skeleton. CT is also widely used for its analgesic effect on bone pain experienced during osteoporosis, although the mechanism for this effect is not clearly understood.
Calcitonin gene related peptide (CGRP) is a neuropeptide whose receptors are widely distributed in the body, including the nervous system and the cardiovascular system. This peptide seems to modulate sensory neurotransmission and is one of the most potent endogenous vasodilatory peptides discovered to date. Reported biological effects for CGRP include: modulation of substance P in inflammation, nicotinic receptor activity at the neuromuscular junction, stimulation of pancreatic enzyme secretion, a reduction of gastric acid secretion, peripheral vasodilation, cardiac acceleration, neuro-modulation, regulation of calcium metabolism, osteogenic stimulation, insulin secretion, an increase in body temperature and a decrease in food intake. Wimalawansa (1997) Crit. Rev. Neurobiol. 11(2-3):167-239. An important role of CGRP is to control blood flow to various organs by its potent vasodilatory actions, as evidenced by a decrease of mean arterial pressure following intravenous administration of α-CGRP. The vasodilatory actions are also supported by recent analysis of homozygous knockout CGRP mice, which demonstrated elevated peripheral vascular resistance and high blood pressure caused by increased peripheral sympathetic activity (Kurihara (2003), Supra). Thus, CGRP appears to elicit vasodilatory effects, hypotensive effects and an increase in heart rate among other actions.
Prolonged infusion of CGRP into patients with congestive cardiac failure has shown a sustained beneficial effect on hemodynamic functions without adverse effects, suggesting a use in heart failure. Other indications of CGRP use include renal failure, acute and chronic coronary artery ischemia, treatment of cardiac arrhythmia, other peripheral vascular disease such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension. Preeclamptic toxemia of pregnancy and preterm labor is also potentially treatable. (Wimalawansa (1997) Supra). Recent therapeutic uses include the use of CGRP antagonists for the treatment of migraine headaches.
Adrenomedullin is almost ubiquitously expressed with many more tissues containing the peptide than not. A published review of ADM details its effects on the cardiovascular system, cellular growth, the central nervous system and the endocrine system, with a range of biological actions including vasodilation, cell growth, regulation of hormone secretion, and natriuresis (Hinson et al. (2000) Endocrine Reviews 21(2): 138-167). Studies in rats, cats, sheep, and man confirm that intravenous infusion of ADM results in potent and sustained hypotension that is comparable to that of CGRP. However, the hypotensive effect of ADM on mean arterial pressure in the anesthetized rat is not inhibited by the CGRP antagonist CGRP8-37 suggesting that this effect is not mediated via CGRP receptors. Acute or chronic administration of human ADM in rats, anesthetized, conscious or hypertensive, results in a significant decrease in total peripheral resistance accompanied by a fall in blood pressure, with a concomitant rise in heart rate, cardiac output and stroke volume.
ADM has also been proposed as an important factor in embryogenesis and differentiation and as an apoptosis survival factor for rat endothelial cells. This is supported by recent mouse ADM knockout studies, in which mice homozygous for loss of the ADM gene demonstrated defective vascular formation during embryogenesis and thus died mid-gestation. It was reported that ADM+/−heterozygous mice had high blood pressure along with susceptibility to tissue injury (Kurihara (2003), Supra.).
ADM affects such endocrine organs as the pituitary, the adrenal gland, reproductive organs and the pancreas. The peptide appears to have a role in inhibiting ACTH release from the pituitary. In the adrenal gland, it appears to affect the secretory activity of the adrenal cortex in both rat and human and it increases adrenal blood flow, acting as a vasodilator in the adrenal vascular bed in intact rats. ADM has been shown to be present throughout the female reproductive tract and plasma levels are elevated in normal pregnancy. Studies in a rat model of preeclampsia show that ADM can reverse hypertension and decrease pup mortality when given to rats during late gestation. Because it did not have a similar effect in animals in early gestation or nonpregnant rats in the preeclampsia model, this suggests that ADM may play an important regulatory role in the utero-placental cardiovascular system. In the pancreas, ADM most likely plays an inhibitory role since it attenuated and delayed insulin response to an oral glucose challenge, resulting in initial elevated glucose levels. ADM can also affect renal function. A bolus administered peripherally can significantly lower mean arterial pressure and raise renal blood flow, glomerular filtration rate and urine flow. In some cases, there is also an increase in Na+ excretion.
ADM also has other peripheral effects on bone and on the lung. For bone, studies have supported a role beyond the cardiovascular system and fluid homeostasis and have demonstrated that ADM acts on fetal and adult rodent osteoblasts to increase cell growth comparable to those of known osteoblast growth factors such as transforming growth factor-β. This is important clinically as one of the major challenges in osteoporosis research is to develop a therapy that increases bone mass via osteoblastic stimulation. In the lung, ADM not only causes pulmonary vasodilation, but also inhibits bronchoconstriction induced by histamine or acetylcholine. Recent studies using aerosolized ADM to treat pulmonary hypertension in a rat model indicate that inhalation treatment of this condition is effective, as evidenced by the fact that mean pulmonary arterial pressure and total pulmonary resistance were markedly lower in rats treated with ADM than in those given saline. This result was achieved without an alteration in systemic arterial pressure or heart rate (Nagaya et al. (2003) Am. J. Physiol. Heart Circ. Physiol. 285:H2125-2131).
A review published by Nicholls et al. (Peptides (2001) 22:1745-1752) summarizes the effects of infusion of ADM. In healthy volunteers, i.v. infusion reduced arterial pressure and stimulated heart rate, cardiac output, plasma levels of cAMP, prolactin, norepinephrine and rennin. In these patients, there was little or no increase in urine volume or sodium excretion observed. In patients with heart failure or chronic renal failure, i.v. ADM had similar effects to those seen in normal subjects and also induced diuresis and natriuresis, depending on the dose administered. Experimental ADM treatment has also been shown to be beneficial in arterial and pulmonary hypertension, septic shock and ischemia/reperfusion injury (Beltowski (2004) Pol. J Pharmacol. 56:5-27). Other indications for ADM treatment include: peripheral vascular disease, subarachnoid hemorrhage, hypertension, preeclamptic toxemia of pregnancy and preterm labor, and osteoporosis.
As the newest member of the Amylin Family of peptides, the biological function of AFP-6 is less well characterized than the members discussed above. However, the expression data obtained from Northern blots of human and mouse tissue is shown in Table 4, in the Example section below, and is consistent with reported data indicating that expression is primarily in the pituitary and gastrointestinal tract. A specific receptor for AFP-6 has not been reported; however, binding studies indicate that AFP-6 binds to all the known receptors of the Amylin Family. AFP-6 has been shown to increase cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competes with labeled CGRP for binding to its receptors in these cells. In published in vivo studies, AFP-6 administration led to blood pressure reduction in both normal and spontaneously hypertensive rats, most likely via interactions with the CRLR/RAMP receptors. In vivo administration in mice led to a suppression of gastric emptying and food intake (Roh et al (2004) J. Biol. Chem. 279(8):7264-7274).
Studies using mutant mice deficient for a CGRP, ADM, or amylin have indicated that, in different systems, CRLR can be important for cardiovascular morphogenesis, sensory neurotransmission, inflammatory reactions, nociceptive behavior, and glucose homeostasis. Thus, the physiological functions of polypeptides in this family are determined by receptor-binding specificity and the tissue expression profiles of individual ligands. AFP-6 appears to be unique in that it binds to all the receptors of the Amylin Family. For example, while amylin binds to the amylin, calcitonin and CGRP receptors with 0.05 nM to 20 nM affinity, it does not bind with very high affinity to the adrenomedullin receptor (several hundred nM affinity). In bovine studies, AFP-6 was shown to bind the adrenomedullin receptor (around 1-5 nM) in addition to having between 3-30 mM binding at the other 3 receptors, giving it amylin, CT, CGRP and/or ADM properties. See also Table 3 herein for receptor binding data.
Amylin Family proteins in general, and AFP-6 polypeptides in particular, have a variety of biological activities that are of use in the treatment or prevention of a variety of diseases, conditions, and disorders. There remains a need to develop such polypeptides, as well as derivatives and analogs thereof, for use in treating and/or preventing the described diseases, conditions, and disorders.